Anne-Marie Koop

6 237 nuclei (blue), black scale bar is equivalent to 20 mm; from the images obtained we determined l) capillaries per cardiomyocytes ratios and m) relative capillary density, density of WT-sham animals was set at 1; n=30 microscopic field/heart, 3 hearts; n) Quantitative real-time PCR analysis of vascular endothelial growth factor receptor 2 (Vegfr2) expression levels of hearts of RV from wildtype (WT) and MHC-miR-199b Tg (MHC-199b) animals, either after sham or PAB surgery. All PCR data are from 5-8 animals per group. Statistical analysis using One-way ANOVA with Tukey’s multiple comparisons test. ∗ p<0.05 versus corresponding control group; #p<0.05 versus corresponding experimental group (error bars are s.e.m.). AsRVcapillaryrarefaction isaphenomenon that leads tomaladaptiveRVremodelling, we assessed the capillary density of the RV in our different experimental groups. Histochemical analysis and respective quantification, as well as determination of vascular endothelial growth factor receptor 2 ( Vegfr2 ) expression levels, reflected lower capillary density in both WT and MHC-199b after PAB, in agreement with similar endoglin levels, suggesting that increased miR-199b levels are not directly associated with RV microvascular remodelling ( figure 3k-n ). Cardiac overexpression of miR-199b increases LV susceptibility to remodelling under RV stress conditions As we have previously shown, miR-199b is upregulated during LV failure and its overexpression increases the sensitivityof the LVto cardiac stress, although indirectly, RV chronic stress can also affect LV remodelling and function. 36-38 Therefore, we also analyzed the changes in molecular profile and morphological adaptation of the LV under conditions of RV pressure overload in both WT and MHC-199b hearts. PAB was able to induce miRNA-199b expression in the LV of WT animals but not in the TG animals ( figure 4a ) when compared to the sham controls. PAB equally increased LV weight indicative of LV hypertrophic growth in the two genotypes ( figure 4b ). Histological analysis revealed increased fibrosis in the LV of TG animals subject to PAB, compared to WT ( figure 4c,d ), but no changes in cardiomyocyte hypertrophy were observed in the left myocardium ( figure 4e,f ). Furthermore, PAB did not induce capillary rarefaction in the LV, independently of the genotype ( figure 4g ). Surprisingly, there was not only a clear upregulation of cardiac stress genes such as nppa , nppb and myh7 ( figure 4h-j ) in the LV of TG mice subjected to PAB, but we also observed increased levels of Rcan 1-4 in the LV of bothWT and MHC-199b after PAB, with a more pronounced effect in the TG animals ( figure 4k ). Congruent to this, left ventricular expression levels of Dyrk1A were reduced upon RV pressure overload, an effect that was more prominent in TG animals ( figure 4l ). RV pressure overload also induced Tgf β expression in the LV with higher levels in the TG mice ( figure 4m ). No changes were observed in Vegfr2 mRNA abundance which is consistent with no changes in LV capillary density ( figure 4n ).